Bifidobacteria are natural inhabitants of the gastrointestinal tract possessing genetic adaptations that enable colonization of this harsh and complex habitat. Bifidobacteria interact with key elements of intestinal functioning and contribute to maintaining homeostasis. Recent scientific progress has demonstrated that bifidobacteria, through strain-dependent interactions with the host may reduce mucosal antigen load, improve the intestinal barrier, and induce regulation of local and systemic immune responses. Due to their recognized benefits to human health bifidobacteria are used as probiotics. Probiotics are “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2001). About a dozen Bifidobacterium strains with clinically documented effects are commercially available. Half of these are Bifidobacterium animalis subsp. lactis strains and the remaining are Bifidobacterium longum subsp. longum, B. longum subsp. infantis, or Bifidobacterium breve strains.
The type strain of Bifidobacterium adolescentis (ATCC15703T) was isolated from the intestine of an adult (Reuter, 1971). Strains of B. adolescentis are frequently detected in the adult human intestinal tract (Turroni et al., 2009).
The intestinal epithelium is the columnar and nonciliated cell layer that covers the small and large intestine. The intestinal epithelial layer constitutes the largest and most important barrier against the external environment and maintaining epithelial integrity is essential to preserving health. The epithelial lining consists of a single layer of epithelial cells covered by layers of mucus produced by specialized goblet cells. Underneath the epithelial cells is the lamina propria containing a variety of immune cells (Gut-associated lymphoid tissue; GALT). Epithelial cells are joined together by cell junctions of which tight junctions (TJ) play a major role in preventing molecules to enter the epithelium between cells.
TJ are responsible for restricting paracellular (between cells) diffusion of proteins, lipids and small solutes. Thus, in a healthy epithelium only water and small molecules (ions) penetrate paracelluarly while transport of larger molecules is regulated by cellular uptake mechanisms. TJ consist of proteins spanning the space between two adjacent intestinal epithelial cells. TJ are dynamic structures that are involved in developmental, physiological and pathological processes. Various stressors may cause weakening of TJ, thus increasing paracellular (un-regulated) transport of molecules into the mucosa. A compromised gut barrier function is characterized by increased permeability of the intestinal mucosa to luminal macromolecules, antigens, and toxins which may cause inflammation, degeneration and/or atrophy of the mucosa. This condition, sometimes referred to as ‘leaky gut syndrome’ is associated with a multitude of symptoms depending on severity. Lipopolysaccharides (LPS) derived from Gram-negative bacteria in the intestine are very potent activators of the immune response. Once the mucosal immune system is activated pro-inflammatory mediators aggravates the opening of TJ resulting in a vicious circle of increasing permeability and inflammation.
Probiotic bacterial strains have been shown to decrease intestinal epithelial permeability, in vitro (Anderson et al., 2010; Karczewski et al., 2010; Liu et al., 2010a, Liu et al., 2010b, Donato et al 2010), in mouse models (Generoso et al., 2010; Liu et al., 2011; Miyauchi et al., 2009), and in humans (Karczewski et al., 2010). Generally good agreement between in vitro and in vivo results has been found.
Mechanisms involved in probiotic improving of barrier function include increased expression of TJ proteins, such as occludin, claudin-1, F11 receptor (F11R), and zona occludens 1 (ZO-1) and 2 (Anderson et al., 2010; Liu et al., 2010a; Liu et al., 2010b; Miyauchi et al., 2009; Ukena et al., 2007. Increased localization of occludin and ZO-1 to the vicinity of TJ structures was found in human biopsies (Karczewski et al., 2010) and in Caco-2 monolayers treated with L. plantarum WCFS1 (Karczewski et al., 2010), or L. rhamnosus LGG (Donato et al., 2010), possibly involving Toll-like (TLR) receptor 2 signaling (Karczewski et al., 2010).
In a neonatal mouse model of necrotizing enterocolitis (NEC) intestinal permeability increases were found to precede NEC, while B. infantis BB-02 administration attenuated intestinal permeability increase, preserved occludin and claudin 2 and 4 localization at TJ, and decreased NEC incidence (Bergmann et al., 2013). The increased intestinal permeability associated with colitis in mice was completely prevented by probiotics (VSL#3) by counterbalancing decreased expression and redistribution of occludin, ZO-1, and claudin-1, -3, -4, and 5 (Mennigen et al., 2009). Proposed bacterial signaling components include the Lactobacillus plantarum surface layer protein (Liu et al., 2010a), and indole (Bansal et al., 2010).
In vivo, barrier function may be measured by various a non-invasive assay methods by administering a bolus of for example CrEDTA or two non-metabolized sugars (e.g. lactulose and mannitol) followed by determining Cr or the ratio of the two sugars in urine, respectively. Mannitol is a monosaccharide and therefore easily absorbed and serves as a marker of transcellular uptake, while the disaccharide lactulose is excluded by the cell lining and thus only slightly absorbed and serves as a marker for mucosal integrity. The lactulose and mannitol test provide integrity information related to only the small intestine, due to bacterial breakdown of the sugars in the large intestine, whereas CrEDTA is more stable and preferentially provides information about the colonic epithelium since this is where the compound is present for the longest time (Arrieta et al., 2006).
Insufficient intestinal barrier function is associated with both intestinal and systemic clinical manifestations. Intestinal permeability has been most extensively studied in the context of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Inflammatory bowel diseases (IBD; Crohn's disease and ulcerative colitis) are characterized by chronic relapsing intestinal inflammation with involvement of both the innate and adaptive immune system (Zhang and Li, 2014). Pro-inflammatory pathways involving cytokine interleukin 23 (IL-23) and T-helper 17 (TH17) cells are elevated in patients with ulcerative colitis and Crohn's disease (Song et al., 2013) which is supported by genetic findings implying an association between gene variants in the IL23R gene and IBD (Beaudoin et al., 2013). The etiology of IBD is unknown, but extensive supporting data of a compromised intestinal barrier function in IBD exists (Geese et al., 2011; Gerova et al., 2011; Odenwald and Turner, 2012). In Crohn's patients with active disease increased intestinal permeability was found (Ukabam et al., 1983) and also 10-20% of healthy relatives to patients with Crohn's disease have increased permeability (Hollander et al., 1986). One theory of IBD pathogenesis suggests that an increased intestinal permeability exposes the underlying GALT to normally excluded agents that results in a self-perpetuating inflammatory process (Poritz et al., 2007). In the dextran sulfate sodium (DSS) colitis mouse model it has been shown that increased intestinal permeability precedes the development of significant intestinal inflammation (Poritz et al., 2007).
Symptoms of irritable bowel syndrome (IBS) include abdominal cramping and pain that is often concurrent with abnormal bowel habits with diarrhea, constipation, or alternating episodes of both. The etiology and pathophysiology of IBS are unknown. Several studies have shown increased intestinal permeability in IBS (Camilleri et al., 2007; Camilleri et al., 2012; Gecse et al., 2011; Martinez et al., 2012; Piche et al., 2009). Increased permeability results from disruption of normal apical expression of TJ proteins claudin-1, ZO-1 and occludin (Camilleri et al., 2012). The increased intestinal permeability is accompanied by persistent low-grade immune activation in the intestine. A previous study found elevated fecal calprotectin in IBS patients (Goepp et al., 2014) indicating elevated inflammation. Also cytokine dysregulation may be involved in the inflammatory process and recent meta-analyses have shown associations between the IL-10 and tumor necrosis factor alpha (TNFα) gene polymorphisms and IBS (Qin et al., 2013; Schmulson et al., 2013; Bashashati et al., 2012). A serum/plasma imbalance in the TNFα cytokine was observed in IBS compared to controls (Bashashati et al. 2014) and in diarrhea predominant IBD patients serum levels of both IL-6 and TNFα were significantly higher compared to controls (Rana et al., 2012). Altogether this suggests an immune displacement towards a more pro-inflammatory stage.
Treatment with probiotic fermented milk (Streptococcus thermophilus, Lactobacillus bulgaricus, L. acidophilus, and B. longum) significantly decreased small intestinal permeability in IBS patients and improved mean global IBS scores (Zeng et al., 2008).
Chronic liver disease is associated with changes in the intestine and liver diseases have been associated with gut microbial changes (Schnabl and Brenner, 2014). These changes in microbial composition may lead to activation of the mucosal immune system via TLR-receptors and Nod like-receptors (NLR) that recognize microbial products followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) activation that initiates immune cell recruitment (Chassaing et al. 2014). Genetically modified animals such as TLR4 mutant mice developed significantly less hepatic fibrosis and hepatic macrophage recruitment after bile duct ligation compared to wild type mice indicating involvement of the TLR4 pathway in development of chronic hepatic diseases (Seki et al. 2007). The connection between cholestatic liver disease and local inflammation in the intestinal lamina propria was shown to be mediated by TLR2-positive monocytes secreting TNFα which was also associated with disruption of the TJ proteins ZO-1 and claudin-4 (Hartmann et al. 2012). Translocation of bacteria or their products, e.g. LPS to mesenteric lymph nodes and extraintestinal sites is common in patients with liver cirrhosis due to increased intestinal permeability (Seo and Shah, 2012), and in patients with chronic liver disease and intestinal bacterial overgrowth with bacterial translocation, disease severity correlated with systemic LPS levels (Lin et al. 1995). In both animal models and chronic liver disease patients, antibiotic treatment improves disease severity by reducing bacterial burden and endotoxemia (Seki et al. 2007; Cirera et al. 2001). However, a leaky gut and translocation of microbial products also occur early in disease and patients with liver disease have a disrupted gut barrier and bacterial products are found in the systemic circulation. Microbial products reach the liver via the portal vein or the lymphatic ducts, where they activate hepatic receptors of the innate immune system (Schnabl, 2013).
Experiments in non-alcoholic fatty liver disease (NAFLD) patients carried out by Miele et al. (2009) strongly suggest that NAFLD is associated with increased gut permeability and small intestinal bacterial overgrowth. Bacterial translocation is correlated with plasma levels of pro-inflammatory cytokines and activation of nitric oxide synthase (Frances et al., 2010), which may cause liver injuries. Thus, reducing bacterial translocation could represent a treatment to alleviate liver diseases.
Decreased bacterial translocation to mesenteric lymph nodes, portal and arterial blood was found in an acute liver injury rat model after treatment with combinations of lactobacilli (L. acidophilus NM1, L. rhamnosus GG, L. plantarum 299v, L. rhamnosus 271, and B. animalis NM2). In addition reduced levels of Enterobacteriaceae (Gram-negative bacteria) were found in cecum and colon. Decreased hepatocellular damage was indicated by lower levels of serum alanine aminotransferase (Adawl et al., 2001). Probiotic treatment not only decreases bacterial translocation, but also reduces endotoxemia caused by endotoxins, mainly LPS derived from Gram-negative bacteria. It seems plausible that endotoxins are important to the development of NAFLD and nonalcoholic steatohepatitis (NASH) via Kuppfer cell stimulation and TNFα production (Osman et al., 2007). Reduced plasma endotoxin levels may be the result of decreasing intestinal permeability.
Reduced concentrations of plasma endotoxin in cirrhosis patients have been found after treatment with two probiotic mixtures (Bifidobacterium, L. acidophilus and Enterococcus [Bifico®], or Bacillus subtilis and Enterococcus faecium [Medilac-s®]) (Wigg et al., 2001), or after treatment with a synbiotic product (Pediococcus pentosaceus 5-33:3, Leuconostoc mesenteroides 32-77:1, L. paracasei subsp. paracasei F19, L. plantarum 2592+bioactive, fermentable fibers; Medipharm), or treatment with the probiotic mixture alone (Zhao et al., 2004). A marginal lowering of endotoxemia was found after treatment with E. coli Nissle 1917 compared to placebo in cirrhotic patients (Liu et al., 2004).
It is well-known that alcohol increases intestinal permeability and this may accelerate the progression of liver disease by increasing portal circulating endotoxin (LPS). Soluble factors from L. rhamnosus LGG was found to reduce the alcohol-induced intestinal permeability increase and endotoxin translocation, and to ameliorate the acute alcohol-induced liver injury in a mouse model (Wang et al., 2012). Improvement of the gut barrier by probiotics is well-documented in vitro, and in vivo. Given the importance of bacterial translocation and endotoxemia to the development of liver diseases it seems likely that probiotics with gut barrier fortifying properties would have a beneficial effect on NAFLD and NASH.
Metabolic disorders (type 2 diabetes and insulin resistance) and obesity are tightly linked to inflammation. Recent evidence suggests an interaction between high-fat diet and bacteria, and the intestinal mucosa may promote small intestine inflammation as an early event in the development of obesity and insulin resistance (Ding and Lund, 2011). Animal studies showed an upregulation in TNFα in ileum in high-fat diet fed mice before weight and fat gain became evident and also the pro-inflammatory pathway of NF-KB was upregulated in ileum and to a lesser degree in colon in high-fat diet fed mice (Ding et al., 2010). In severely obese children fecal calprotectin was increased in 47% of the patients, whereas rectal nitric acid was pathologically high in 88% of the obese children and in 100% of the diabetic patients supporting the hypothesis that distal intestinal inflammation is involved in obesity and diabetes (Spagnuolo et al., 2010). In a study in obese women, gene expression of pro-inflammatory pathways was dramatically down-regulated after a diet-induced weight loss of an average of 10%, accompanied by reduction in TNFα, IL-1β, IL-8 and monocyte chemotactic protein 1 and macrophage infiltration (Pendyala et al., 2011). Altogether, these data implies that the intestine in obese subjects and diabetic patients have an increased inflammatory status compared to healthy subjects and that this may proceed the development of weight gain and glucose/insulin imbalances. Germ-free mice are protected against the metabolic complications of exposure to a high-fat/high-refined sugar ‘Western’ diet. Translocated bacterial LPS has been identified as a triggering factor of low-grade, chronic inflammation, termed ‘metabolic endotoxemia’ (Cani et al., 2007). According to this model, LPS is released from lysing Gram-negative bacteria in the intestine and translocates across the epithelium when the barrier is compromised, e.g. as a consequence of a high-fat containing diet. The increased levels of plasma LPS (2-3-fold) causes a slightly increased, but persistent, inflammatory tone that triggers weight gain and insulin resistance (Cani et al., 2007). Increased circulating levels of LPS and markers of intestinal permeability (zonulin) are found in patients with type 2 diabetes (Hawkesworth et al., 2012; Jayashree et al., 2014) as well as type 1 diabetes (de Kort et al., 2011; Vaarala et al., 2008). Zonulin upregulation, i.e. increased intestinal permeability seems to precede the onset of type 1 diabetes (Sapone et al., 2006). Colonization of the intestines with bifidobacteria enhances intestinal barrier function through increasing ZO-1 and occludin expression, and significantly and positively improves glucose tolerance, glucose-induced insulin secretion and normalizes the inflammatory tone (Cani et al., 2007).
Metabolic endotoxemia due to loss of intestinal barrier integrity activates TLR4-mediated inflammation and induce oxidative stress which is associated with increased cardiovascular risk and mortality. Increased translocation of LPS through the intestinal barrier causes higher circulating levels of LPS that promotes atherosclerosis (Neves et al., 2013). Markers of systemic inflammation such as circulating LPS is elevated in patients with chronic infections and are strong predictors of increased atherosclerotic risk (Kiechl et al., 2001).
Key elements of autoimmune diseases are adaptive immunity and an imbalance between TH1 and TH2 immune responses. In neonates microbial antigens can induce a TH1 immune response that offsets the normally dominant TH2 immune response. A TH1 immune response is characteristic of autoimmune and inflammatory diseases. Recently, a compromised intestinal barrier has been proposed to be involved in the development of autoimmune diseases (Fasano and Shea-Donohue, 2005). According to this hypothesis there are three key elements in the pathogenesis of autoimmune diseases.                1. A miscommunication between innate and adaptive immunity,        2. Continuous stimulation by nonself-antigen (environmental triggers) perpetuates the process.        3. A loss of protective function of mucosal barriers that interact with the environment (gastrointestinal and lung mucosa).        
Pathology of celiac disease is an example. Early in the development of celiac disease TJ are opened and intestinal tissue damage results. Gliadin triggers the zonulin innate immunity pathway in a MyD88-dependent way that initiates opening of TJ and induces a pro-inflammatory (TH1) response in the intestinal mucosa. Once gliadin (gluten) is removed from the diet, serum zonulin levels decrease, the intestine resumes its baseline barrier function, autoantibody titers are normalized, and the autoimmune process shuts off (Fasano and Shea-Donohue, 2005; Fasano, 2012).
Several other autoimmune diseases, including type 1 diabetes, multiple sclerosis and rheumatoid arthritis, are characterized by increased intestinal permeability that allow the passage of antigens from the intestinal microbiota, challenging the immune system to produce an immune response that can target any organ or tissue (by molecular mimicry) in genetically predisposed individuals (Fasano, 2012). Furthermore, the immune system of particularly the small intestine has been recognized to induce tolerogenic responses to for example food antigens or commensals that may be involved in the development of autoimmune diseases. The small intestine was acknowledged to redirect and control pro-inflammatory TH17 cells (Esplugues et al. 2011) and it has been proposed that probiotic bacteria may act by modulating intestinal immune system and thus dampen disease development and severity in animal models of rheumatoid arthritis and multiple sclerosis (So et al., 2008; Kwon et al., 2013)
Germ-free mice have an exaggerated hypothalamic-pituitary-adrenal reaction to stress compared to conventional mice, which can be reversed by monoassociation with B. infantis suggesting a cross-talk between gut bacteria and the brain (Sudo et al., 2004), increased gut permeability, bacterial translocation and activation of the TLR4 pathway have been implicated as a link between psychological disorders and somatic diseases, including mood disorders, cognitive disorders, and chronic fatigue syndrome. Elevated expression of markers of the TLR4 pathway was found in patients diagnosed with major depressive disorder, accompanying increased bacterial translocation across the intestinal barrier (Keri et al., 2014). Translocated Gram-negative gut bacteria and LPS activate immune cells to elicit IgA and IgM responses that cause progressive amplifications of immune pathways associated with neuroinflammation and neuroprogression and with the onset of melancholic symptoms, e.g. anhedonia, anorexia, weight loss, psychomotor retardation, anxiety, and fatigue (Macs et al., 2012).
Transepithelial Electrical Resistance (TER)
The barrier properties of epithelial cell monolayers are determined to a large extent by TJ located in the intercellular space where they form a seal between the apical and basolateral membrane domain and regulate paracellular passage of molecules. The barrier function is not static but can be deliberately modulated by exposure to specific stimuli. The resulting dynamics of TJ network can be conveniently followed by measuring the transepithelial electrical resistance (TER). Caco-2 is a well-established cell line derived from human colon adenocarcinoma which is commonly used as an intestinal permeability model. When fully differentiated Caco-2 monolayers form TJ restricting transfer of ions and, thus, produce an electrical resistance across the monolayer.
BD™ Cytometric Bead Array (CBA)
The mucosal-associated lymphoid tissues lining the human gastrointestinal tract contain a network of immune cells. Dendritic cells (DCs) govern the balance between immunity and tolerance by sampling of intestinal contents and initiating appropriate immune responses to luminal antigens through pattern recognition receptor signaling, cytokine secretion, and their ability to migrate and present antigen to naïve T cells in draining lymph nodes. At homeostasis, DCs in the intestinal mucosa are conditioned by commensal microorganisms to promote proliferation of Foxp3+ regulatory T cells (Tregs), strong producers of anti-inflammatory IL-10 contributing to intestinal tolerance. Luminal antigens translocating through the epithelial call layer bind to pattern recognition receptors expressed on DCs and activate signaling pathways resulting in production and secretion of a wide range of chemokines and cytokines with distinct inflammatory effects. In this context, DC secretion of inflammatory cytokines such as TNFα, IL-1b, IL-6, and IL-12 is central for acute, innate inflammatory responses involving attraction of neutrophils and macrophages to the site of infection. In addition, DCs are central players in the regulation of adaptive immune responses, thus, DC modulation toward an IL-10 secreting phenotype contributes to induction of Treg responses promoting intestinal tolerance (Smith et al., 2014). Multiplexed immunoassays based on the principles of flow cytometry allow for simultaneous determination of numerous soluble proteins in very small sample volumes. The combination of high throughput and impressive accuracy, sensitivity, and reproducibility make these experimental techniques highly relevant for screening purposes where rapid quantification of multiple compounds is critical.
The DSS Colitis Model
The rodent Dextran Sulfate Sodium (DSS) colitis model features uncontrolled colonic inflammation. In many ways it resembles IBD, including ulcerative colitis (UC) and Crohn's disease, for which worldwide incidence and prevalence has been shown to increase (Molodecky et al., 2012). The underlying pathophysiological mechanisms of DSS colitis include initial disruption of intestinal barrier function followed by inflammation and crypt loss (Cooper et al., 1993; Iwaya et al., 2012; Poritz et al., 2007). Disease symptoms in DSS colitis correspond to what is observed in human UC, including body weight loss, diarrhea and fecal blood loss (Herias et al., 2005). The exact mechanism whereby DSS induces colitis is not elucidated, however, it has been recognized that DSS associates with medium-chain-length fatty acids present in the colonic lumen and form vesicles capable of fusing with the colonocytes membranes which may cause activation of inflammatory signaling pathways in the cytoplasm (Laroui et al., 2012). Also, recent findings indicate that thickness of the inner colonic mucus layer, normally devoid of bacteria, is decreased and becomes permeable to bacteria only 15 minutes after DSS exposure. Within 12 hours after DSS exposure bacterial interaction with the epithelial layer was observed which could activate inflammation (Johansson et al., 2010). As in healthy rodents, bacteria are clearly separated from the epithelial colonic layer in healthy humans, whereas in ulcerative colitis patients with acute inflammation bacteria penetrate the inner mucus layer (Johansson et al., 2014), indicating common pathology between the DSS-induced colitis model and human inflammatory GI disorders.
One of the early events of DSS-induced pathology is loss of tight junction ZO-1 and increased intestinal permeability, preceding intestinal inflammation (Poritz et al., 2007), which could indicate that the DSS disrupts intestinal barrier function, allowing for penetration of toxins, antigens and whole or fractions of bacteria which fuel inflammation. This also corresponds well with human findings where the intestinal permeability is compromised along with marked downregulation in TJ genes during intestinal inflamed conditions (Koltun et al., 1998; Gassler et al., 2001). Interestingly, down-regulation of TJ genes like ZO-1, claudin-1, JAM, beta-catenin and occludin in colonic mucosa areas actively transmigrating neutrophils was seen in ulcerative colitis patients (Kucharzik et al., 2001) suggesting a close connection between the disruption in TJs and inflammation.
Bifidobacteria, lactobacilli and mixtures have shown efficacy in DSS-induced colitis in mice and rats (Chen et al., 2009; Kim et al., 2010; Geier et al., 2007; Mennigen et al., 2009). Different probiotic modes of action have been proposed involving strengthening of the intestinal epithelial barrier and modulating inflammatory pathways such as cytokine signaling. For example, Lactobacillus reuteri inhibited bacterial translocation from the intestine to the mesenteric lymph nodes in addition to the disease activity index (Dicksved et al., 2012) which could suggest increased barrier function as a part of the disease severity dampening mechanism. Also, E. coli Nissle 1917 was shown to dampen DSS-induced colitis by strengthening of intestinal permeability and 13 protein expression such as ZO-1 (Ukena et al., 2007). Although this mode of action has not been directly verified in humans thus far E. coli Nissle 1917 has been reported as being efficacious in preventing ulcerative colitis relapse (Kruis et al., 1997; Kruis et al., 2004) indicating similarities in mechanisms between rodents and humans.
Modulations of inflammatory pathways during DSS-induced colitis by the use of probiotics were also shown to effectively inhibit disease severity. Yao and co-workers transfected a B. longum with an IL-10 containing plasmid and dosed the bacteria to mice exposed to DSS. The transfected bacteria alleviated the colitis symptoms by downregulating the NF-KB pathway that would otherwise lead to production of various pro-inflammatory cytokines (Yao et al., 2011). Miyauchi and others on the other hand showed that B. longum subsp. infantis was capable of reducing colitis severity by suppressing the expression of type 1 helper T (TH1) and IL-17 producing helper T (TH17)-specific cytokines in colonic tissue (Miyauchi et al., 2013).